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Tube, Micropipettor, Vortex mixer, 37℃ thermostat water bath, Spectrophotometer (340 nm)
This kit can be used for detection of MDH activity in animal tissues, plant tissues and culture cells.
MDH has close links with various important ways of plant metabolism. It plays an important role in the process of Mal / OAA (malic acid/oxaloacetic acid) and Mal / Asp (malic acid / aspartic acid) transporting material and energy; In photorespiration, MDH provides NAD+ for the oxidation of Gly (glycine); In mitochondria, MDH is one of the regulatory enzymes that decide the running speed of TCA (tricarboxylic acid cycle); In cytosol, MDH is related to the branch of pyruvic acid. Therefore, MDH system is not only a good system to research the regionalization and regulation of enzyme, but also provides convenience for researching the relation among every organelle and many development problems.
1. Put ultraviolet spectrophotometer at 340 nm wavelength with 0.5 cm optical path quartz colorimetric utensil and set to zero with double-distilled water (Prepare two quartz colorimetric utensil. One for setting zero, the other for measuring).
2. Pre-heat working solution at 37℃ for more than 3 min.
3. Add 50 μL sample into tubes with corresponding numbers. Take 1 mL working solution and pour it into tube rapidly. Mix immediately and reckon time. (Take 50 μL double-distilled water and add 1 mL working solution into blank tube. Other steps are the same as the measurement.)
4. Pour into quartz colorimetric utensil. Make colorimetric reaction with ultraviolet spectrophotometer at 340 nm wavelength. Measure the OD value at 20th second (A1); measure the OD value again at 80th second (A2).
5. Work out the difference of two OD values (△A = A1 - A2).
Note: Prepare one or two blank tubes (ODBlank is very stable).
If △ASample / min<0.05, you should increase the concentration of sample. Otherwise detection result will be influenced.
If △ASample / min>0.3, you should dilute the concentration of sample, then test. Otherwise detection result will be influenced.
Take normal control sample to do pre-experiment about the optimum concentration of this sample before batch inspection.